Throughout this specification the use of the word “inventor” in singular form may be taken as reference to one (singular) inventor or more than one (plural) inventor of the present invention.
It is to be appreciated that any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the present invention. Further, the discussion throughout this specification comes about due to the realisation of the inventor and/or the identification of certain related art problems by the inventor. Moreover, any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventor's knowledge and experience and, accordingly, any such discussion should not be taken as an admission that any of the material forms part of the prior art base or the common general knowledge in the relevant art in Australia, or elsewhere, on or before the priority date of the disclosure and claims herein.
Attenuated Total Reflection Infrared (ATR-IR) Spectroscopy
Spectroscopy is the branch of science devoted to discovering the chemical composition of materials by examining the interaction of electromagnetic radiation with the material. Infrared (IR) spectroscopy relates primarily to the absorption of energy by molecular vibrations having wavelengths in the infrared segment of the electromagnetic spectrum, that is energy of wave number between 200 and 4000 cm−1. Raman spectroscopy relates to the inelastic scattering of monochromatic light giving wavelength shifts that depend on the molecular vibrations, having typically wave number shifts between 20 and 4000 cm−1.
ATR is a sampling technique that can be used in conjunction with IR. ATR spectroscopy offers the advantages of being potentially portable, it is inexpensive and thus has become a very powerful tool in the analysis of biological cells and tissues. ATR also allows samples to be examined directly in the solid or liquid state without further preparation, and compared with transmission-IR, the path length into the sample is shorter, avoiding strong attenuation of the IR signal in highly absorbing media such as aqueous solutions.
In use, the sample is put in contact with the surface of a crystal having a higher refractive index than the sample. A beam of IR light is passed through the ATR crystal in such a way that it reflects at least once off the internal surface in contact with the sample. This reflection forms an evanescent wave which extends into the sample. The penetration depth into the sample depends on the wavelength of light, the angle of incidence and the indices of refraction for the ATR crystal and the medium being probed. The number of reflections may be varied. The beam is then collected by a detector as it exits the crystal.
Malaria
Malaria is a mosquito borne disease caused by parasitic protozoans of the genus Plasmodium. Five species of Plasmodium can infect humans—P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi—but the vast majority of deaths are caused by P. falciparum. P. falciparum causes up to 1.2 million fatalities per annum. Accurate and early diagnosis followed by the immediate treatment of the infection is essential to reduce mortality and prevent overuse of antimalarial drugs.
New technologies to diagnose malaria must be cost effective and have high sensitivity and be able to detect circulating stages of the malaria parasite namely the ring and gametocyte forms because these are the only stages present in peripheral blood circulation.
The current suite of malarial diagnostics in clinical use include: (i) optical microscopy of thick blood films, (ii) Rapid Diagnostic Tests (RDTs) based on the detection of antigens specific to P. falciparum, (iii) gene amplification techniques such as polymerase chain reaction (PCR) and (iv) serological detection tests using antibodies such as immunofluorescence (IFA) and enzyme-linked immunosorbent assay (ELISA).
Each method has its own advantages and disadvantages. For example, optical microscopy requires preparation of blood smear samples using reagents and is based on visual assessment of the morphology of blood cells. The method is inherently subjective and requires experienced microscopists to make diagnosis.
Polymerase chain reaction (PCR) is considered the most sensitive and specific method, but has the drawbacks of being time consuming, technically sophisticated, expensive, and requiring a PCR machine, and is thus not suitable for malaria diagnosis in remote areas. Malaria RDTs, which are based on capture of parasite antigens by monoclonal antibodies incorporated into a test strip, are easy to use but are unable to quantify parasitemia.
A review of existing methods indicates that the examination of stained blood smears by light microscopy remains the method of choice for malaria diagnosis because it is inexpensive and has good sensitivity (5-10 parasites/μl blood). However, it is labor-intensive, lengthy, and more importantly, requires skilled and experienced microscopists, and is increasingly burdensome as malaria rates decline with most smears examined being negative.
During the course of its life the malaria parasite transgresses through several developmental stages including a sexual and an asexual reproductive pathway. The sexual or progeny phase, which occurs within the gut of female Anopheles mosquito, produces numerous infectious forms known as sporozoites that are transferred to the mosquito salivary glands and injected into the human host during a blood meal.
Sporozoites that enter a blood vessel move to the liver and invade hepatocytes where they develop into schizonts each containing tens of thousands of merozoites. The merozoites are subsequently released and invade the erythrocytes initiating the intraerythrocytic asexual phase of the life cycle. The merozoites grow and divide in the food vacuole and progress through three distinct morphological phases known as the ring, trophozoite and schizont stages (FIG. 1).
Mature stage parasites adhere to the vascular endothelium and thus only ring stage parasites are observed in blood smears. The schizonts burst, releasing the merozoites and the intraerythocytic cycle continues. Instead of replicating, some merozoites in the erythrocytes develop into sexual forms of the parasite, called male and female gametocytes, that are capable of undergoing transmission to mosquitoes.
Early stage gametocytes sequester away from the peripheral circulation but late stage gametocytes are present in blood smears, and gametocyte carriage underpins endemicity of disease. The detection of the rings in peripheral blood is critical for early diagnosis and treatment. The detection of low levels of gametocytes in asymptomatic long-term malaria carriers is critical to efforts to eradicate malaria.
During the intraerythrocytic stages of the parasites life cycle P. falciparum endocytoses packets of host cell cytoplasm, catabolizes the lipids and hemoglobin and in the process releases free heme, which is toxic to the organism. The malaria parasite has evolved a detoxification pathway that uses the lipid by-products to catalyze the sequestration of free heme into an insoluble pigment known as hemozoin (Hz). Hence Hz is a disposal product formed from the digestion of blood by malaria parasites (and some other blood feeding parasites).
Synchrotron powder diffraction analyses have shown that crystals of Hz (and its synthetic equivalent β-hematin) are composed of a repeating array of iron-carboxylate interacting heme dimers, stabilized by hydrogen bonding and π-π interactions.
Vibrational spectroscopic techniques have been used extensively in understanding the molecular and electronic structure of β-hematin and Hz; however, the use of vibrational spectroscopy for malaria diagnostics has not been fully exploited. Raman imaging microscopy has been explored as a potential non-subjective method to diagnose malaria parasites based on the strong scattering from the Hz pigment. (Wood et al, Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics, Analyst 2009, 134. 1119-1125). While the technique has shown potential to detect ring forms of the parasite the time taken to record an image is on the order of several hours and therefore not suitable for the clinical environment.
Efforts have also been made to investigate the potential of synchrotron Fourier Transform Infrared (FTIR) in combination with Principal Component Analysis (PCA) to differentiate between intraerythrocytic stages of the parasite life cycle based on the molecular signatures of Hz and specific lipids (Webster et al. Discriminating the Intraerythrocytic Lifecycle Stages of the Malaria Parasite Using Synchrotron FT-IR Microspectroscopy and an Artificial Neural Network. Analytical Chemistry 2009, 81. 2516-2524). Webster et al found that as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage there is an increase in absorbance and shifting of specific lipid bands.
This work demonstrated the potential of using FTIR spectroscopy as a diagnostic tool for malaria but clearly a synchrotron-based method is not suitable for routine laboratory use.
In particular, malaria detection methods of the prior art have focussed on detection of Hz. However, one of the principal problems with relying solely on the detection of Hz is that early forms of the malaria parasite (the ring stage) have very small amounts of Hz. Thus, many Raman methods of the prior art can optimally detect trophozoites which have large amounts of Hz, however this suffers the drawback that trophozoites are not generally found in peripheral blood.